1) The objective of the laboratory was to transform E. coli bacteria with a gene from jellyfish that codes for Green Fluorescent Protein (GFP).
2) The bacteria samples were transformed using a CaCI2 solution and incubated on agar plates containing the transformed bacteria colonies.
3) Examination of the agar plates under UV light showed expression of GFP in some colonies, indicating successful genetic transformation. SDS-PAGE electrophoresis identified the GFP protein band at 27 kD in transformed bacteria samples.
Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. Mammalian cell culture technology has become a major field in modern biotechnology; mammalian cell culture refers to the cells of a mammalian, isolated from specific tissues (i.e. skin, liver, glands, etc.) and further cultivated and reproduced in an artificial medium. Cell culture technology is currently playing a major role in toxicity testing, cancer research, virology, genetic engineering, and gene therapy.
OBJECTIVE:
To observe the transfection of CHO and HEK cells with GFP
To observe the recombinant GFP using Western Blotting
To purify the transfected HEK and CHO cells using AKTA Pure Purification
Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. Mammalian cell culture technology has become a major field in modern biotechnology; mammalian cell culture refers to the cells of a mammalian, isolated from specific tissues (i.e. skin, liver, glands, etc.) and further cultivated and reproduced in an artificial medium. Cell culture technology is currently playing a major role in toxicity testing, cancer research, virology, genetic engineering, and gene therapy.
OBJECTIVE:
To observe the transfection of CHO and HEK cells with GFP
To observe the recombinant GFP using Western Blotting
To purify the transfected HEK and CHO cells using AKTA Pure Purification
1. University of Puerto Rico<br />Cayey Campus<br />Jessica Díaz Rivera<br />Prof. Elena Gonzalez<br />RISE Program<br />Laboratory #7: pGLO Genetic Transformation and SDS Page Extension<br /> A gene is a piece of DNA, which provides the instructional codes for proteins. Genetic transformation means “change caused by genes”, and involves the insertion of a gene into an organism in order to change the organism’s trait. The objective of this laboratory was to transform bacteria, in this case E. coli, with a gene that is from a jellyfish and codes for Green Fluorescent Protein (GFP). We used a transformation solution (CaCI2) in each sample. To maintain the area aseptic we used the burner to work with bacteria. Later we used a sterile loop to pick up a single colony of bacteria from the starter plate. After incubating all of the samples,. we put 100 micro liters of each sample in the appropriate agar plate. In the next laboratory, we qualitatively examined the GFP on agar plates. Then we identified the protein in polyacrylamide gel (SDS-PAGE electrophoresis). We did the SDS-PAGE in the .75 ml electrophoresis, but we could not put all the samples into the gel because of the density of the solution. The gel had ten lanes, 5 with UV illumination downstream process and the other 5 with Coomassie staining. It also contained, four samples: white and green heat and white and green no heat. As a result, we saw the GFP protein in the 27 kD in the gel. We also learned how to use the BIO RAD Molecular Imager Gel Doc machine to take a photo of the gel.<br />
